Bi-specific monoclonal antibody (specific for both CD3 and CD11B) therapeutic drug

ABSTRACT

The present invention relates to the treatment of immune system abnormalities that can be found in lethal human cancers and also in the progressive Human Immunodeficiency Virus Type 1 (HIV-1) infections and provides medicaments to correct abnormalities in a subject with cancer or HIV-1-infected subjects, in order to allow the immune system to fight the cancer or HIV-1 infections. The present invention also discloses multivalent polypeptides which specifically bind to and enable destruction and/or inactivation of immune cells that have CD11b and CD3 on their surface, therefore dissipating the deleterious effects of the CD11b + T cells.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a divisional and claims the benefit of priorityunder 35 U.S.C. §120 of U.S. patent application Ser. No. 11/821,127,filed Jun. 22, 2007, currently pending, which claims the benefit ofpriority under 35 U.S.C. §119(e) of U.S. Provisional Application No.60/834,816, filed Jul. 29, 2006, the contents of which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to immunotherapy and, more specifically,to multivalent polypeptides directed to CD3 and CD11b cell surfacemarkers, which markers are present on a subpopulation ofimmunosuppressing T cells, for therapeutic and diagnostic and prognosticuse in cancer and infectious disorders.

2. Background Information

Patients with Chronic Lymphocytic Leukemia (CLL), usually a “B” cellcancer, commonly have abnormally large populations of certain kinds ofnon-cancerous “T” cells in the blood which have many CD11b molecules ontheir surface. Similarly, patients with progressive debilitating HIV-1infections also have abnormally large populations of T cells with CD11bmolecules on their surface, which normally comprise only a smallpercentage of the T cells in the blood of healthy people.

Evidence indicates that these T cells with CD11b on their surface (i.e.,“CD11b⁺”) can interfere with or suppress the body's immune responses,and thus may have deleterious effects on one's ability to fight offcancer or HIV-1 infection. It appears that many types of human cancerand HIV-1 infection may elicit increased production of these CD11b⁺Tcells as a way of evading the immune response of the host.

SUMMARY OF THE INVENTION

The present invention relates to the treatment of immune systemabnormalities that can be found in lethal human cancers and also in theprogressive Human Immunodeficiency Virus Type 1 (HIV-1) infections.Specifically, the present invention provides medicaments to correctabnormalities in a subject with cancer or who is HIV-1-infected, inorder to allow the immune system to better fight cancer or HIV-1infections.

The present invention relates to multivalent polypeptides whichspecifically bind to and enable destruction and/or inactivation ofimmune cells that have CD11b and CD3 on their surface, thereforedissipating the deleterious effects of the CD11b⁺T cells.

In one embodiment, an isolated multivalent polypeptide whichspecifically binds to CD3 and CD11b surface markers is disclosed.

In one aspect, the antibody is a bispecific, trispecific, tetravalent,hexavalent, octavalent, or decavalent antibody. In another aspect, themultivalent polypeptide is conjugated to a cytotoxic agent.

In another embodiment, a method for the treatment of an immunologicaldisorder in a subject is disclosed, including administering to thesubject, in an amount effective for the treatment, a pharmaceuticalcomposition comprising (a) at least one multivalent polypeptide that (i)immunospecifically binds CD3 and CD11b surface markers and (ii) exerts acytostatic or cytotoxic effect on a subpopulation of T-cell; and (b) apharmaceutically acceptable carrier. In one aspect, the treatment may bein vitro, ex vivo, or by administration of the multivalent polypeptideintra- or peritumorally, where intra- or peritumoral administrationinduces infiltration by immunoeffector cells.

In one aspect, the immunological disorder includes, but is not limitedto, cancer, breast cancer, skin cancer, bone cancer, prostate cancer,liver cancer, lung cancer, brain cancer, cancer of the larynx,gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neuraltissue, head and neck, colon, stomach, bronchi, kidneys, basal cellcarcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma,veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lungtumor, gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell leukemia,adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosalneuromas, intestinal ganglioneuromas, hyperplastic corneal nerve tumor,Wilm's tumor, seminoma, ovarian tumor, cervical dysplasia and in situcarcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignantcarcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma,osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor,adenocarcinoma, leukemias, lymphomas, malignant melanomas, epidermoidcarcinomas, and other carcinomas and sarcomas.

In a related aspect, the cancer is chronic lymphocytic leukemia (CLL).

In another aspect, the immunological disorder is a viral infection,where the viral infection includes, but is not limited to, Epstein-Barrvirus, human immunodeficiency virus, human T leukemia virus, hepatitis Bvirus, or measles virus.

In one embodiment, a kit is disclosed, including a multivalentpolypeptide which specifically binds to CD3 and CD11b surface markers, alabel, and instruction for using the multivalent polypeptide.

In another embodiment, a method for diagnosing an immunological disordercharacterized by an increase in a circulating subpopulation of T cellsbearing CD3 and CD11b cell surface markers is disclosed, includingcontacting a biological sample with a multivalent polypeptide whichspecifically binds to CD3 and CD11b surface markers and detectingbinding. In one aspect, the detecting step comprises an immunoassay.

In another aspect, detecting of cells is by multicolor flowimmunocytometry, fluorescence activated cell sorting (FACS), magneticactivated cell sorting (MACS), or digital image microscopy.

In one embodiment, a method of isolating a T cell subpopulation from asample is disclosed, where the T cell subpopulation suppresses theimmune response, including contacting the sample with a multivalentpolypeptide which specifically binds to CD3 and CD11b surface markersunder conditions suitable for the formation of an antibody-T cellcomplex, isolating a population of CD3⁺/CD11b⁺T cells from the sample,and substantially separating the isolated cells.

In a related aspect, the cells are substantially separated by a methodincluding but not limited to, fluorescence activated cell sorting (FACS)and magnetic activated cell sorting (MACS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustration of an example bispecific antibody of thepresent invention.

FIG. 2 shows a list of T lymphocyte abnormalities commonly found inuntreated cases of B-cell CLL.

FIG. 3 shows a list of acquired immune defects in patients with cancer.

FIG. 4 shows a life-table analysis for two groups of patients, where onegroup has a T-cell count of 160 or less and the other group has a T-cellcount of greater than 160.

DETAILED DESCRIPTION OF THE INVENTION

Before the present composition, methods, and isolation methodologies aredescribed, it is to be understood that this invention is not limited toparticular compositions, methods, and experimental conditions described,as such compositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “apolypeptide” includes one or more polypeptides, and/or compositions ofthe type described herein which will become apparent to those personsskilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, as it will be understood thatmodifications and variations are encompassed within the spirit and scopeof the instant disclosure.

“Bispecific antibody” and “bispecific antibodies,” also known asbifunctional antibodies, intends antibodies that recognize two differentantigens by virtue of possessing at least one first antigen combiningsite specific for a first antigen or hapten, and at least one secondantigen combining site specific for a second antigen or hapten. Suchantibodies can be produced by recombinant DNA methods or include, butare not limited to, antibodies produced chemically by methods known inthe art. Chemically created bispecific antibodies that have been reducedand reformed so as to retain their bivalent characteristics andantibodies that have been chemically coupled so that they have at leasttwo antigen recognition sites for each antigen. Bispecific antibodiesinclude all antibodies or conjugates of antibodies, or polymeric formsof antibodies which are capable of recognizing two different antigens.Bispecific antibodies include antibodies that have been reduced andreformed so as to retain their bivalent characteristics and toantibodies that have been chemically coupled so that they can haveseveral antigen recognition sites for each antigen.

In one embodiment, an isolated multivalent polypeptide whichspecifically binds to CD3 and CD11b surface markers is disclosed.

In one aspect, the antibody is a bispecific antibody. In another aspect,the multivalent polypeptide is conjugated to a cytotoxic agent.

As used herein the term “effector cell population” intends a cellpopulation which comprises at least one T cell. An effector cellpopulation can be obtained from a starting cell population from whichantigen-specific T cells are enriched.

The terms “cell,” and “cells,” and “cell population,” usedinterchangeably, intend one or more mammalian cells. The term includesprogeny of a cell or cell population. Those skilled in the art willrecognize that “cells” include progeny of a single cell, and the progenycan not necessarily be completely identical (in morphology or of totalDNA complement) to the original parent cell due to natural, accidental,or deliberate mutation and/or change.

The terms “T lymphocyte,” “T cell,”. “T cells,” and “T cell population,”used interchangeably, intends a cell or cells which display on theirsurface one or more antigens characteristic of T cells, such as, forexample, CD3 and CD11b. The term includes progeny of a T cell or T cellpopulation. A “T lymphocyte” or “T cell” is a cell which expresses CD3on its cell surface and a T cell antigen receptor (TCR) capable ofrecognizing antigen when displayed on the surface of autologous cells,or any antigen-presenting matrix, together with one or more MHCmolecules or, one or more non-classical MHC molecules. The term “Tcells” as used herein denotes any T cells known in the art, forinstance, lymphocytes that are phenotypically CD3⁺, i.e., express CD3 onthe cell surface, typically detected using an anti-CD3 monoclonalantibody in combination with a suitable labeling technique. The T cellsenriched by the methods of this invention are generally CD3⁺. The Tcells enriched by the methods of this invention are also positive forCD11b.

In one embodiment, a method of isolating a T cell subpopulation from asample is disclosed, where the T cell subpopulation suppresses theimmune response, including contacting the sample with a multivalentpolypeptide which specifically binds to CD3 and CD11b surface markersunder conditions suitable for the formation of an antibody-T cellcomplex, isolating a population of CD3⁺/CD11b⁺T cells from the sample,and substantially separating the isolated cells.

The term “substantially enriched” or “substantially isolated” as usedherein, indicates that a cell population is at least about 50-fold, morepreferably at least about 500-fold, and even more preferably at leastabout 5000-fold or more enriched from an original mixed cell populationcomprising the desired cell population.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, humans, farm animals,sport animals, and pets.

An “effective amount” is an amount sufficient to effect beneficial ordesired clinical results. An effective amount can be administered in oneor more administrations. For purposes of this invention, an effectiveamount of multivalent polypeptide is an amount that is sufficient todiagnose, palliate, ameliorate, stabilize, reverse, slow or delay theprogression of the disease state.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, preventing spread (i.e., metastasis) ofdisease, delay or slowing of disease progression, amelioration orpalliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment.

In another embodiment, a method for the treatment of an immunologicaldisorder in a subject is disclosed, including administering to thesubject, in an amount effective for the treatment, a pharmaceuticalcomposition including (a) at least one multivalent polypeptide that (i)immunospecifically binds CD3 and CD11b surface markers and (ii) exerts acytostatic or cytotoxic effect on a subpopulation of T-cell; and (b) apharmaceutically acceptable carrier.

In one aspect, the immunological disorder includes, but is not limitedto, cancer, breast cancer, skin cancer, bone cancer, prostate cancer,liver cancer, lung cancer, brain cancer, cancer of the larynx,gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neuraltissue, head and neck, colon, stomach, bronchi, kidneys, basal cellcarcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma,veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lungtumor, gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell leukemia,adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosalneuromas, intestinal ganglioneuromas, hyperplastic corneal nerve tumor,Wilm's tumor, seminoma, ovarian tumor, cervical dysplasia and in situcarcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignantcarcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma,osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor,adenocarcinoma, leukemias, lymphomas, malignant melanomas, epidermoidcarcinomas, and other carcinomas and sarcomas.

In a related aspect, the cancer is chronic lymphocytic leukemia (CLL).

In another aspect, the immunological disorder is a viral infection,where the viral infection includes, but is not limited to Epstein-Barrvirus, human immunodeficiency virus, human T leukemia virus, hepatitis Bvirus, or measles virus.

The effector cell population can be subjected to one or more separationprotocols based on the expression of cell surface markers. For example,the cells can be subjected to positive selection on the basis ofexpression of one or more cell surface polypeptides, including, but notlimited to, “cluster of differentiation” cell surface markers such asCD2, CD3, CD4, CD8, TCR, CD45, CD45RO, CD45RA, CD11b, CD26, CD27, CD28,CD29, CD30, CD31, CD40L; other markers associated with lymphocyteactivation, such as the lymphocyte activation gene 3 product (LAG3),signaling lymphocyte activation molecule (SLAM), T1/ST2; chemokinereceptors such as CCR3, CCR4, CXCR3, CCR5, homing receptors such asCD62L, CD44, CLA, CD146, a4(37, aE37; activation markers such as CD25,CD69 and OX40; and lipoglycans presented by CD1. The effector cellpopulation can be subjected to negative selection for depletion of non-Tcells and/or particular T cell subsets. Negative selection can beperformed on the basis of cell surface expression of a variety ofmolecules, including, but not limited to, B cell markers such as CD19,and CD20; monocyte marker CD14; the NK cell marker CD56.

Generally, antibodies suitable for practicing the methods of the presentinvention immunospecifically bind CD3 and CD11b. Antibodies suitable forpracticing the methods of the invention are preferably monoclonal andmultivalent, and may be human, humanized or chimeric antibodies,comprising single chain antibodies, Fab fragments, F(ab′) fragments,fragments produced by a Fab expression library, and/or binding fragmentsof any of the above. The term “antibody,” as used herein, refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain at least twoantigen binding sites that immunospecifically bind CD3 and CD11b. Theimmunoglobulin molecules of the invention can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule. In certain embodimentsof the invention, the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera V_(L) or V_(H) domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH₁, CH₂, CH₃ and CL domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH₁, CH₂, CH₃ and CL domains. Preferably,the antibodies are human, murine (e.g., mouse and rat), donkey, sheep,rabbit, goal, guinea pig, camelid, horse, or chicken. As used herein,‘human’ antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries, from human B cells, or from animals transgenicfor one or more human immunoglobulins.

In one embodiment, the antibody is a bispecific antibody as illustratedin FIG. 1. In one aspect, the antibody may comprise human IgG1, with abinding site A (top V_(L)/V_(H) region), capable of binding to a CD3molecule, and binding site B (lower corresponding region) capable ofbinding to a CD11b molecule.

The antibodies suitable for practicing the methods of the presentinvention may be bispecific, trispecific or of greater multispecificity.Further, the antibodies of the present invention have low risk oftoxicity against granulocyte (neutrophil), NK cells, and CD4⁺ cells asbystander cells.

Multivalent antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e., bispecific antibodies, BsAbs), antibodies withadditional specificities such as trispecific antibodies are encompassedby this expression when used herein.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al, EMBOJ., 10:3655-3659 (1991).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. Such interfaces may comprise at least a part of the CH₃ domainof an antibody constant domain. In this method, one or more small aminoacid side chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoet-hylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J Exp. Med, 175: 217-225 (1992) describe theproduction of a fully humanized bispecific antibody F(ab′)₂ molecule.Each Fab′ fragment was separately secreted from E. coli and subjected todirected chemical coupling in vitro to form the bispecific antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe ErbB2 receptor and normal human T cells, as well as trigger thelytic activity of human cytotoxic lymphocytes against human breast tumortargets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kos-telny et al., J. Immunol, 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).Alternatively, the antibodies can be “linear antibodies” as described inZapata et al. Protein Eng. 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H1)-V_(H)-C_(H1)) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Multivalent antibodies may be specific for different epitopes of CD3 andCD11b, including, for example, that the multivalent antibodies may bindto one or more of the epitopes present on either CD3 or CD11b.Multivalent antibodies, including bispecific and trispecific antibodies,useful for practicing the present invention are antibodies thatimmunospecifically bind to both CD3 and CD11b, and may bind one of moreadditional lymphocyte surface receptors or receptor complexes, such asan immunoglobulin gene superfamily member, a TNF receptor superfamilymember, an integrin, a cytokine receptor, a chemokine receptor, a majorhistocompatibility protein, a lectin (C-type, S-type, or I-type), or acomplement control protein.

Antibodies useful in the present methods may be described or specifiedin terms of the particular CDRs they comprise. The invention encompassesthe use of an antibody or derivative thereof comprising a heavy or lightchain variable domain, said variable domain comprising (a) a set ofthree CDRs, and (b) a set of four framework regions, and in which saidantibody or derivative thereof immunospecifically binds CD3 and CD11b.

Within the context of the present invention, antibodies are understoodto include monoclonal antibodies and polyclonal antibodies, antibodyfragments (e.g., Fab and F(ab′)₂), chimeric antibodies bifunctional orbispecific antibodies and tetrameric antibody complexes. Antibodies areunderstood to be reactive against a selected antigen on the surface of aT cell if they bind with an appropriate affinity (association constant),e.g. greater than or equal to 10⁷ M⁻¹. Additionally, antibodies that maybe used in the methods of the present invention may also be described orspecified in terms of their binding affinities include those with adissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, or 10⁻¹⁵ M.

Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forthe whole antibodies. For example, F(ab′)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab′)₂ fragment can betreated to reduce disulfide bridges to produce Fab′ fragments

The invention also contemplates chimeric antibody derivatives, i.e.,antibody molecules that combine a non-human animal variable region and ahuman constant region. Chimeric antibody molecules can include, forexample, the antigen binding domain from an antibody of a mouse, rat, orother species, with human constant regions. A variety of approaches formaking chimeric antibodies have been described and can be used to makechimeric antibodies containing the immunoglobulin variable region whichrecognizes the selected antigens on the surface of differentiated cellsor tumor cells. See, for example, Morrison et al., 1985; Proc. Natl.Acad. Sci. U.S.A. 81,6851; Takeda et al., 1985, Nature 314:452; Cabillyet al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397;Tanaguchi et al., European Patent Publication EP171496; European PatentPublication 0173494, United Kingdom patent GB 2177096B.

Chemical conjugation is based on the use of homo- and heterobifunctionalreagents with E-amino groups or hinge region thiol groups.Homobifunctional reagents such as 5,5′-Dithiobis(2-nitrobenzoic acid)(DNTB) generate disulfide bonds between the two Fabs, and0-phenylenedimaleimide (O-PDM) generate thioether bonds between the twoFabs (Brenner et al., 1985, Glennie et al., 1987). Heterobifunctionalreagents such as N-succinimidyl-3-(2-pyridylditio) propionate (SPDP)combine exposed amino groups of antibodies and Fab fragments, regardlessof class or isotype (Van Dijk et al., 1989).

The antibodies of the invention, i.e., antibodies that are useful fortreating immunological disorders, include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody from bindingto CD3/CD11b. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of turicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

The antibodies that may be used in the treatment of immunologicaldisorders may be generated by any suitable method known in the art.Polyclonal antibodies to CD3/CD11b can be produced by various procedureswell known in the art. For example, CD3/CD11b can be administered tovarious host animals including, but not limited to, rabbits, mice, rats,etc. to induce the production of sera containing polyclonal antibodiesspecific for the protein. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.,1988); Hammer-ling, et al., in: Monoclonal Antibodies and T-CellHybrido-mas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with CD3/CD11b or a cellexpressing CD3/CD11b or a fragment or derivative thereof. Once an immuneresponse is detected, e.g., antibodies specific for CD3/CD11b aredetected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of bindingCD3/CD11b and exerting a cytotoxic or cytostatic effect on activatedlymphocytes. Ascites fluid, which generally contains high levels ofantibodies, can be generated by injecting mice with positive hybridomaclones.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thevariable region, the light chain constant region and the CH₁ domain ofthe heavy chain.

For example, antibodies useful in the methods of the present inventioncan also be generated using various phage display methods known in theart. In phage display methods, functional antibody domains are displayedon the surface of phage particles which carry the nucleic acid sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g. human or murine). In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the nucleic acid sequences encoding them. Inparticular, DNA sequences encoding V_(H) and V_(L) domains are amplifiedfrom animal cDNA libraries (e.g., human or murine cDNA libraries oflymphoid tissues). The DNA encoding the V_(H) and V_(L) domains arerecombined together with an scFv linker by PCR and cloned into aphagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13 binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Phage expressing an antigen bindingdomain that binds to CD3/CD11b or portions thereof can be selected oridentified with antigen e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Examples of phage display methodsthat can be used to make the antibodies of the present invention includethose disclosed in Brinkman et al, 1995, J. Immunol. Methods 182:41-50;Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al,1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18;Burton et al., 1994, Advances in Immunology, 191-280; PCT ApplicationNo. PCT/GB91/01134; PCT Publications WO 90/02809; WO 91/10737; WO92/01047; WO 92/18619; WO 93/1 1236; WO 95/15982; WO 95/20401; and U.S.Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908;5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;5,658,727; 5,733,743 and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al, BioTechniques 1992,12(6):864-869; and Sawai et al, 1995, AJRI 34:26-34; and Better et al.,1988, Science 240:1041-1043.

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., 1991, Methods in Enzymology 203:46-88; Shu etal., 1993, PNAS 90:7995-7999; and Skerra et al., 1988, Science240:1038-1040. For some uses, including in vivo use of antibodies inhumans and in vitro proliferation or cytotoxicity assays, it ispreferable to use chimeric, humanized, or human antibodies. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science, 1985,229:1202; Oi et al, 1986, Bio-Techniques 4:214; Gillies et al., 1989, J.Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and4,816,397. Humanized antibodies are antibody, molecules from non-humanspecies antibodies that bind the desired antigen having one or more CDRsfrom the non-human species and framework and constant regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., 1988, Nature 332:323. Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology, 1991,28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; Roguska. et al, 1994, PNAS 91:969-973), and chainshuffling (U.S. Pat. No. 5,565,332).

Completely human antibodies are particularly desirable for thetherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

Human antibodies can also be produced using transgenic mice whichexpress human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells. The mouseheavy and light chain immunoglobulin genes may be renderednon-functional separately or simultaneously with the introduction ofhuman immunoglobulin loci by homologous recombination. In particular,homozygous deletion of the JH region prevents endogenous antibodyproduction. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized in the normal fashion witha selected antigen, e.g., all or a portion of CD3 and CD 11b. Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see, Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93.For a detailed discussion of this technology for producing humanantibodies and human monoclonal antibodies and protocols for producingsuch antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047;WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, companiessuch as Abgenix, Inc. (Freemont, Calif.) and Medarex (Princeton, N.J.)can be engaged to provide human antibodies directed against a selectedantigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., 1994, Bio/technology12:899-903).

Further, antibodies to CD3/CD11b can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” proteins of the invention usingtechniques well known to those skilled in the art. (See, e.g. Greenspan& Bona, 1989, FASEB J. 7(5):437-444; and Nissinoff. 1991, J. Immunol.147(8):2429-243S). Fab fragments of such anti-idiotypes can be used intherapeutic regimens to elicit an individual's own immune responseagainst CD3/CD11b present on activated lymphocytes.

Aside from the antibodies specifically identified above, the skilledpractitioner could generate polyclonal antibodies directed against anantigen of interest.

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the antigen to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoy-bean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxy-ysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with the original amountof antigen or conjugate in Freund's complete adjuvant by subcutaneousinjection at multiple sites. Seven to 14 days later the animals are bledand the serum is assayed for antibody titer. Animals are boosted untilthe titer plateaus. Preferably, the animal is boosted with the conjugateof the same antigen, but conjugated to a different protein and/orthrough a different cross-linking reagent. Conjugates also can be madein recombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-1 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, Protein A-Sepharose, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. Preferably theProtein A chromatography procedure described herein is used.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

In a further embodiment, monoclonal antibodies can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al. Nature,352:624-628 (1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991)describe the isolation of murine and human antibodies, respectively,using phage libraries. Subsequent publications describe the productionof high affinity (nM range) human antibodies by chain shuffling (Markset al, Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection a in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalhybridoma techniques for isolation of monoclonal antibodies.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185.

Immunoadhesins may also be used according to the methods of the presentinvention. The simplest and most straightforward immunoadhesin designcombines the binding domain(s) of the adhesin (e.g. the extracellulardomain (ECD) of a receptor) with the hinge and Fc regions of animmunoglobulin heavy chain. Ordinarily, when preparing theimmunoadhesins of the present invention, nucleic acid encoding thebinding domain of the adhesin will be fused C-terminally to nucleic acidencoding the N-terminus of an immunoglobulin constant domain sequence,however N-terminal fusions are also possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, CH₂ and CH₃ domains of the constantregion of an immunoglobulin heavy chain. Fusions are also made to theC-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH₁ of the heavy chain or the corresponding region ofthe light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion, or bindingcharacteristics of the immunoadhesin.

In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc domain of immunoglobulin Gj (IgGj). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and CH₂ and CH₃ or (b)the CH₁, hinge, CH₂ and CH₃ domains, of an IgG heavy chain.

For bispecific immunoadhesins, the immunoadhesins are assembled asmultimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multivalent form in serum.In the case of multimer, each of the four units may be the same ordifferent.

Various exemplary assembled immunoadhesins within the scope herein areschematically diagrammed below:

-   (a) AC_(L)-AC_(L);-   (b) AC_(H)-(AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), or    V_(L)C_(L)-AC_(H));-   (c) AC_(L)-AC_(H)-(AC_(L)-AC_(H3), AC_(L)-V_(H)C_(H),    V_(L)C_(L)-AC_(H), or V_(L)C_(L)-V_(H)C_(H))-   (d) AC_(L)-V_(H)C_(H)-(AC_(H), or AC_(L)-V_(H)C_(H) or    V_(L)C_(L)-AC_(H));-   (e) V_(L)C_(L)-AC_(H)-(AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H)); and-   (f) (A-Y)_(n)-(V_(L)C_(L)-V_(H)C_(H))₂, wherein each A represents    identical or different adhesin amino acid sequences:-   V_(L) is an immunoglobulin light chain variable domain;-   V_(H) is an immunoglobulin heavy chain variable domain;-   C_(L) is an immunoglobulin light chain constant domain;-   C_(H) is an immunoglobulin heavy chain constant domain;-   n is an integer greater than 1;-   Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed tobe present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmuno-globulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the CH₂ domain, or between the CH₂ and CH₃domains. Similar constructs have been reported by Hoogenboom, et al.,Mol. Immunol. 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anadhesin-immunoglobulin heavy chain fusion polypeptide, or directly fusedto the adhesin. In the former case, DNA encoding an immunoglobulin lightchain is typically coexpressed with the DNA encoding theadhesin-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immuno-globulin-like structure comprising twodisulfide-linked immunoglobulin heavy chain-light chain pairs. Methodssuitable for the preparation of such structures are, for example,disclosed in U.S. Pat. No. 4,816,567, issued 28 Mar. 1989.

Immunoadhesins are most conveniently constructed by fusing the cDNAsequence encoding the adhesin portion in-frame to an immunoglobulin cDNAsequence. However, fusion to genomic immunoglobulin fragments can alsobe used (see, e.g. Aruffo et al., Cell 61:1303-1313 (1990); andStamenkovic et al., Cell 66:1133-1144 (1991)). The latter type of fusionrequires the presence of Ig regulatory sequences for expression. cDNAsencoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and theimmunoglobulin parts of the immunoadhesin are inserted in tandem into aplasmid vector that directs efficient expression in the chosen hostcells.

Analysis of the cell population and cell sorting based upon the presenceof the label can be accomplished by a number of techniques known in theart. Cells can be analyzed or sorted by, for example, flow cytometry orFACS. These techniques allow the analysis and sorting according to oneor more parameters of the cells. Usually one or multiple secretionparameters can be analyzed simultaneously in combination with othermeasurable parameters of the cell, including, but not limited to, celltype, cell surface markers, DNA content, etc. The data can be analyzedand cells sorted using any formula or combination of the measuredparameters. Cell sorting and cell analysis methods are known in the artand are described in, for example, The Handbook of ExperimentalImmunology, Volumes 1 to 4, (D. N. Weir, editor); Flow Cytometry CellSorting (A. Radbruch, editor, Springer Verlag, 1992); and CellSeparation Methods and Applications (D. Recktenwald and A. Radbruch,eds., 1997) Marcel Dekker, Inc. N.Y. Cells can also be analyzed usingmicroscopy techniques including, for example, laser scanning microscopy,fluorescence microscopy; techniques such as these can also be used incombination with image analysis systems. Other methods for cell sortinginclude, for example, panning and separation using affinity techniques,including those techniques using solid supports such as plates, beadsand columns.

Some methods for cell sorting utilize magnetic separations, and some ofthese methods utilize magnetic beads. Different magnetic beads areavailable from a number of sources, including for example, Dynal(Norway), Advanced Magnetics (Cambridge, Mass., U.S.A.), Immun-con(Philadelphia, U.S.A.), Immunotec (Marseilles, France), and Miltenyi Biotec GmbH (Germany).

Preferred magnetic labeling methods include colloidal superparamagneticparticles in a size range of 5 to 200 nm, preferably in a size of 10 to100 nm. These magnetic particles allow a quantitative magnetic labelingof cells, thus the amount of coupled magnetic label is proportional tothe amount of bound product, and the magnetic separation methods aresensitive to different amounts of product secretion. Colloidal particleswith various specificities are known in the art, and are available, forexample, through Miltenyi Biotec GmbH. The use of immunospecificfluorescent or magnetic liposomes can also be used for quantitativelabeling of captured product. In these cases, the liposomes containmagnetic material and/or fluorescent dyes conjugated with antibody ontheir surfaces, and magnetic separation is used to allow optimalseparation between nonproducing, low producing, and high producingcells.

The magnetic separation can be accomplished with high efficiency bycombining a second force to the attractive magnetic force, causing aseparation based upon the different strengths of the two opposed forces.Typical opposed forces are, for example, forces induced by magneticfluids mixed in the separation medium in the magnetic separationchamber, gravity, and viscous forces induced by flow speed of mediumrelative to the cell. Any magnetic separation method, preferablymagnetic separation methods allowing quantitative separation will beused. It is also contemplated that different separation methods can becombined, for example, magnetic cell sorting can be combined with FACS,to increase the separation quality or to allow sorting by multipleparameters.

Preferred techniques include high gradient magnetic separation (HGMS), aprocedure for selectively retaining magnetic materials in a chamber orcolumn disposed in a magnetic field. In one application of thistechnique the product is labeled by attaching it to a magnetic particle.The attachment is generally through association of the product with alabel moiety which is conjugated to a coating on the magnetic particlewhich provides a functional group for the conjugation. The capturedproduct thus coupled to a magnetic “label”, is suspended in a fluidwhich is then applied to the chamber. In the presence of a magneticgradient supplied across the chamber, the magnetically labeled targetcell is retained in the chamber; if the chamber contains a matrix, itbecomes associated with the matrix. Cells which do not have or have onlya low amount of magnetic labels pass through the chamber.

The retained cells can then be eluted by changing the strength of, or byeliminating, the magnetic field or by introducing a magnetic fluid. Theselectivity for a captured product is supplied by the label moietyconjugated either directly or indirectly to the magnetic particle or byusing a primary antibody and a magnetic particle recognizing the primaryantibody. The chamber across which the magnetic field is applied isoften provided with a matrix of a material of suitable magneticsusceptibility to induce a high magnetic field gradient locally in thecamber in volumes close to the surface of the matrix. This permits theretention of fairly weakly magnetized particles. Publications describinga variety of HGMS systems are known in the art, and include, forexample, U.S. Pat. Nos. 4,452,773, 4,230,685, PCT applicationWO85/04330, U.S. Pat. No. 4,770,183, and PCT/EP89/01602; systems arealso described in U.S. Pat. Nos. 5,411,863; 5,543,289; 5,385,707; and5,693,539.

In addition, in other embodiments the processes include labeling thecells that contain the product captured by the capture moiety, if any.Other embodiments can also include analyzing the cell population todetect labeled cells, if any, and if desired, sorting the labeled cells,if any.

The present invention further provides diagnostic methods for detectingantigen-specific T cells. These include methods for analyzing apopulation of cells enriched for T cells to identify or enumerateantigen-specific T cells, as well as methods of determining adistribution of antigen-specific T cells.

Methods for analyzing a population of cells enriched in T cells toidentify or enumerate antigen-specific T cells relative to other cellsin the population, comprise the steps of labeling the cells by themethods of the present invention; labeling the cells and detecting theamount of label. Such methods are useful, for example, in determiningthe proportion of a cell population that is specific for a givenantigen. The method can be used to provide information regarding theimmune status of an individual, including assessing an immune responseto allergens, a tumor or virus, or evaluating the proportion of cells inan individual that are self reactive so as to detect or monitorautoimmune diseases.

The present invention provides methods of treatment of a disease orcondition related to a population of antigen-specific T cells, using themultivalent polypeptide of the invention.

Treatment methods include those in which an antigen-specific T cellpopulation is identified, and in an individual; those in which apopulation of antigen-specific T cells is identified anddiminished/reduced in vitro before the T-cell population is reintroducedinto an individual; those in which a population of antigen-specific Tcells is identified and eliminated from a population of cells to beintroduced into an individual; ex vivo genetic modification prior toadministration; and selection of antigen-specific T cells according toCD3/CD11b expression.

In one embodiment, a kit is disclosed, including a multivalentpolypeptide which specifically binds to CD3 and CD11b surface markers, alabel, and instruction for using the multivalent polypeptide. In oneaspect, the components of the kit may be used to predict theeffectiveness of the treatment of a subject administered the multivalentpolypeptide of the present invention, where the subject has animmunological disorder.

The kit can also be formulated to include the following: all thereagents are preferably placed in a single vial to which the cells areadded. At least one antibody which is bispecific for a particular cellsurface structure and, optionally, at least one label moiety

Optionally, the kit can include physiologically acceptable buffer. Suchbuffers are known in the art and include, but are not limited to, PBSwith and without BSA, isotonic saline, cell culture media and anyspecial medium required by the particular cell type. Buffers can be usedthat reduce cross-labeling and increase the local product concentrationaround the cells. Buffers can include agents for increasing viscosity ordecreasing permeability. Suitable agents are described herein. Theviscosity of the medium can be reduced before analysis by any methodknown in the art including, but not limited to, dissolution in aphysiologically acceptable buffer, dissolving heat, EDTA, and enzymes.In the absence of added medium, cells already suspended in a medium canbe directly added to the vial. Suitable cell suspensions include but arenot limited to cell lines and biological samples. Biological samplesinclude, but are not limited to, blood, urine and plasma.

Additional label moieties such as antibodies (magnetically orfluorescently labeled) can also be present, including, but not limitedto anti-cell surface marker antibodies to identify cell types, propidiumiodide to label dead cells, and magnetic beads to label certain celltypes.

In one embodiment, all materials can be placed in a single containersuch as a vial and the cell sample added. The contents are incubated toallow secretion of a product and subsequent capture of the product andbinding of the label moiety to the product. The cells which havesecreted and bound product can then be separated and/or analyzed basedon the presence, absence or amount of the captured product. Separationcan be done by any of the methods known in the art, including, but notlimited to, simple dilution, erythrocyte lysis, centrifugation-washingstep, magnetic separation, FACS and Ficoll separation. The analysis ofthe cells can be performed by a variety of methods, including, but notlimited to, FACS, image analysis, cytological labeling, and immunoassay.

The pharmaceutical compositions of the present invention may be in theform of a sterile injectable aqueous or oleagenous suspension. Thissuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents which havebeen mentioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable (pharmaceutically acceptable) diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

In some aspects, double therapeutic agents may be used comprisingmultivalent polypeptides which deliver toxins or radionuclides,including, but not limited to, P³², P³³, Sc⁴⁷, Cu⁶⁴, Cu⁶⁷, As⁷⁷, Y⁹⁰,PH¹⁰⁵, Pd¹⁰⁹, Ag¹¹¹, I¹²⁵, Pr¹⁴³, Sm¹⁵³, Tb¹⁶¹, Lu¹⁷⁷, Re¹⁸⁶, Re¹⁸⁸,Re¹⁸⁹, Ir¹⁹⁴, Au¹⁹⁹, Pb²¹², and Bi²¹³.

In other aspects, the chemotherapeutic agents may be used including, butnot limited to, adriamycin, doxorubicin, epirubicin, 5-fluorouracil,cytosine arabinoside, cyclophosphamide, thiotepa, busulfan, cytoxin,paclitaxel, doxetaxel, toxotere, methotrexate, cisplatin, melphalan,vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, fludarabine,cladaribine, mitoxantrone, vincristine, vinorelbine, carboplatin,teniposide, daunomycin, caminomycin, aminopterin, dactinomycin,mitomycins, esperamicins, 5-FU, 6-thioguanine, 6-mercaptopurine,actinomycin D, VP-16, chlorambucil, melphalan, or a combination thereof.

In one aspect, a toxin may be used including, but not limited to,diphtheria A chain, a nonbinding active fragment of diphtheria toxin, anonbinding active fragment of cholera toxin, a nonbinding activefragment of botulin toxin, Pseudomonas aeruginosa exotoxin A chain,ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins PAPI,PAPII, PAP-S, Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, saporin, mitogellin, restrictocin,phenomycin, enomycin, tricothecenes, calicheamicins, maytansinoids,palytoxin, CC1065, or a combination thereof. Further, such toxin mayalso possess a radioactive moiety, including, but not limited to thoserecited above.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular subject may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the host undergoing therapy.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES

Generation of Single Chain Antibody Fragments from Monoclonal Antibodiesto CD3 and to CD11b.

The techniques described in this Example can be used to generate asingle chain antibody fragment (ScFv) of either the anti-CD3 or anti-CD11b monoclonal antibodies.

Both the V_(H) and V_(L) region of the antibodies are amplified by PCR,followed by a second assembly PCR to connect both regions. Four primerscan be designed. The first contains restriction site for cloningpurposes followed by a degenerated sequence annealing to the 5′ V_(H)region. The second contains a degenerate sequence for the 3′ part of theV_(H) region followed by a sequence encoding a ((Gly)₄Ser)₃ (SEQ IDNO: 1) linker and the 5′ part of the V_(L) regions. The third is adegenerated primer having homology with the 5′ part of the V_(L) region,while the last primer contains an appropriate restriction site andanneals to the 3′ part of the V_(L) region.

As a template for this PCR reaction, one can use a plasmid containingthe V_(H) or V_(L) regions of the antibody of interest. The cDNAobtained in this PCR step is gel purified and used in an assembly PCRresulting in the linkage of the V region through the ((Gly)₄Ser)₃ (SEQID NO: 1) linker. Subsequently the single chain construct obtained isdigested with the appropriate restriction enzymes, followed by ligationinto an expression vector. The ligation is transformed in DH5a andplated on LB plates. By sequencing of several clones, a correct ScFvclone is found.

Construction of Bispecific Diabody Molecules Capable of Binding to CD4and CD11b.

Bispecific bivalent molecules can be generated by shortening theflexible linker sequence in the anti-CD3 ScFv and in the anti-CD11bScFv, from fifteen residues to five ((Gly)₄Ser)₃ (SEQ ID NO: 1) and bycross-pairing the variable heavy and light chain domains from the twosingle chain Fv fragments with the different antigen recognition. Theconstruction is preferably performed in three steps. The light chainvariable fragments are exchanged in the ScFv constructs from an anti-CD3ScFv and an anti-CD11b ScFv by using restriction enzyme sites located inthe 5′-end and just outside the 3′-part of the light chain variablegene. In the following step, the 15-residue linker of the chimericconstruct V_(H)-a CD3/15AA-linker/V_(L)-a CD11b is replaced by the 5residue linker ((Gly)₄Ser)₃ (SEQ ID NO: 1) by using sites located in the3′-part of V_(H) and the 5′-part of V_(L). Finally, a chimeric cassetteis combined in an appropriate vector, containing a bi-cistronicexpression cassette. A diabody-producing clone containing bothScFv-cassettes is identified and used for expression of the recombinantdiabody molecule.

Example 1 Evidence for Low Risk of Cellular Toxicity for Bystander Cells

Subjects

Normal subjects and a consecutive group of consenting patients atScripps Clinic and Scripps Memorial Hospital-LaJolla, who were diagnosedwith B-CLL were recruited according to an Institutional Review Board[IRB]-approved protocol. Blood specimens were analyzed in a blindedfashion. For leukemic patients, extensive immunophenotyping was done oneach specimen to confirm the diagnosis of B-CLL.

Flow Cytometry

Flow cytometry was performed with FACScan, Ortho-Cytotron, orFACSCalibur instruments, used interchangeably based on availability atEsoterix Oncology. Whole blood preparations were stained and fixed priorto cytometry according to the manufacturer's instructions(Becton-Dickenson) with the exception that red blood cell (RBC) lysiswas done with ammonium chloride-based reagent.

Cytometry Computer Software

Software capable of high resolution multidimensional data analysis wasused (see, e.g., Bjork et al., Clin App Immunol Rev (2002) 2:141-154).The software has both manual and automated population analysis features,and results can be easily displayed and cross-checked with currentlymarketed software that generates 2-dimensional dot plots (e.g., suchsoftware for 2 dimensional dot plots may be obtained from PurdueUniversity Cytometry Laboratory (PUCL) website, hosted by J. PaulRobinson, Director PUCL, Bindley Bioscience Center, Purdue University,West Lafayette, Ind.).

Experimental Data

Patients with B-cell CLL (B-CLL) have a virtual constellation ofassociated T cell disorders (FIG. 2) of unknown etiology. Theseimpairments are similar to those found in blood mononuclear cells ofuntreated B-CLL cases, untreated GM cases, lung cancer cases, coloncancer cases, and breast cancer cases, including: impaired responses torecall antigens, and reduced lymphocyte proliferation reactions toallogenic stimuli and to mitogens such as phytohemagglutinin (PHA) (FIG.3).

A cross sectional study of 23 cases of B-CLL, representing all Raistages, revealed a high prevalence of expanded subsets of CD3⁺/CD11b⁺cells among the CD8⁺ T lymphocytes in the blood. Abnormally largesubsets of these cells were found in 8 of 16 early state (Rai 0 or 1)and 5 of 7 advanced-stage (Rai 2 to 4) cases, some of whom were onchemotherapy at the time of blood drawing. Another trend discovered wasthe ratio of CD28⁺CD8⁺T cells (Tc) to putative immune suppressor T cells(i.e., CD3⁺/CD11b⁺T cells (Ts), or Tc/Ts, tended to be lower in B-CLLcases (mean 1.6, range 0.4-3.8) compared to normal subjects (mean 13,range 1.1-100).

In was observed that seven of the eight cases with abnormally high Tscounts (180 or greater) experienced a CLL-related event during theprospective study period (EFS=12.5%). The eighth case was 14 monthspost-research cytometry, and had no evidence of significant diseaseprogression. In contrast, five of five cases with Ts counts of 160 orless have remained event free (EFS=100%). FIG. 4 illustrates alife-table analysis of the two groups, with median follow-up of 30months, in which the difference in the probability of EFS isstatistically significant (P=0.01). In essence, irrespective of anyB-cell related parameter, the absolute count of putative Ts appeared toindependently correlate with elements of disease progression in theearly stages of these B-CLL cases.

Thirteen cases of B-CLL patients (samples were obtained from J. Moore,Philadelphia) were studied based on progression of their disease (themedian medical follow-up of the updated data is 49 months afterexperimental cytometry and 60 months after diagnosis): among 6 caseswhich continue to have non-progressive disease, the mean absolute countof CD11b⁺/CD3⁺T-cells was 161 (range 91-338, SD=89), while thecorresponding absolute counts among 7 cases with progressive disease was507 (range 180-932; SD=251). Alternatively, these cases could be dividedinto two groups based on absolute CD11b⁺/CD3⁺ counts: 8 cases had highcounts (>2 SD above the mean for normal healthy controls) and 5 caseshad normal counts. Although the age sex, Rai stage, and absolutelymphocyte counts were not significantly different in the two groups,there was a statistically significant difference in progression-freesurvival between the two groups: 7 of the 8 cases with high CD11b⁺/CD3⁺Tcell counts (180 or greater) had disease progression, while 5 of 5 withnormal counts remain progression-free and asymptomatic (p=0.01).

A predominant majority of granulocytes (i.e., neutrophils) and theirprecursors express CD11b on the cell surface. Thus, any drug/agenttargeting only the CD11b molecule will be expected to have significant“bystander” toxicity on the granulocyte cell lineage. Similarly, naturalkiller (NK) lymphocytes (i.e., CD56⁺ lymphocytes) also express CD 11b ontheir cell surface, and would also become “bystander” targets of aCD11b-focused therapeutic strategy. However, there is no consensus onwhether a significant percentage of T-helper cells (i.e., CD4⁺ cells)express CD11b.

If a therapeutic intervention is restricted to only those cells thatco-express CD11b and CD3, it is important to establish what percentageof granulocytes, NK cells, and T-helper cells co-express these twomarkers.

In 4 of 6 cases of Chronic Lymphocytic Leukemia (CLL), less than 1% ofgranulocytes co-express CD11b and CD3, while in the other 2 cases 9.5%and 7% of granulocytes co-express these molecules (see, Table 1).

TABLE 1 % of Neutrophils (Granulocytes) that Express CD3 on theirSurfaces: 65 Cases of CLL Case % Expression Case 1: C. B. <1% Case 2: M.A. <1% Case 3: R. E. <1% Case 4: M. M. <1% Case 5: J. W. 9.50%    Case6: H. G.   7%

In 4 cases of CLL, 1.3%, 4%, 6%, and 25% of NK cells express CD3 ontheir surface (see, Table 2).

TABLE 2 % of NK cells (CD 56+ lymphocytes) that Express CD3 on TheirSurfaces: 4 Cases of CLL. Case % Expression Case 1: J. G. 1.30%   Case2: G. B. 4% Case 3: H. S. 6% Case 4: B. H. 25% 

In 10 cases of CLL, the percentage of T helper cells that co-expressCD11b ranges from 2.5% to 17%, with a mean of 5.6%. This data shows thata small percent of CD3⁺ and CD11b⁺ cells are T-helper (i.e. CD4⁺) cells,so that the percentage of CD11b+/CD3⁺ lymphocytes is approximately thesame as the percentage of CD8⁺/CD11b⁺/CD3⁺ lymphocytes (see, Table 3).

TABLE 3 Summary of Flow Cytometry Data on Blood Lymphocytes Subsets inB-CLL Study Subjects* CD3⁺ CD8⁺ NK T CD8⁺ CD8⁺ CD56⁺ CD3⁺ Lymphs CD19⁺Cells Cells T4/T8 CD28⁻ CD11b⁺ CD11b⁺ CD11b⁺ Cases Normals (34 (28-39)13 (11-16) 14 (10-19) 72 (67-76) 1.2 (1.0-1.5) (≦16) (≦16) (≦16) T4 1M.S. 37 24 3 12 1.50 55 45 14 6 2 G.D. 44 30 6 21 2.60 35 27 18 2.5 3D.G. 64 43 9 20 0.57 76 24 3 6 4 A.S. 18 4 2 53 0.39 39 23 1 2.5 5 K.H.78 64 1 6 0.66 63 62 25 17 6 M.R.B. 61 43 1 19 5.00 27 18 4 5 7 L.M. 6145 2 19 0.73 36 22 1 2.7 8 S.B.D. 48 43 2 6 1.90 20 24 20 4 9 W.P. 49 378 11 1.00 76 52 9 3.4 10 W.S. 64 61 1 4 2.80 16 30 5 8 *These cases area random series of cases, without knowledge of age, stage of disease, ortreatment. Proportions of total lymphoid cells and CD19⁺ cells areexpressed as percentages of all events in the listmode file. NK (CD56⁺)and T(CD3⁺) cells are expressed as percentages of lymphocytes. Thesubsets of CD11b⁺/CD8⁺ T cells are shown as the percentage of all CD8+ Tcell. “%” normal values, if known, are in parentheses.

This data shows that a very small percentage of CD3⁺/CD11b⁺ cells areT-helper (i.e., CD4⁺) cells. Thus, CD8⁺/CD11b⁺ are equivalent toCD8⁺/CD3⁺ populations.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method for the treatment of B-cell chronic lymphocytic leukemia(B-CLL) in a subject, comprising administering to the subject, in anamount effective for the treatment, a pharmaceutical compositioncomprising (a) at least one multivalent polypeptide that (i)immunospecifically binds CD3 and CD11b surface markers simultaneouslyexpressed on the surface of a regulatory T cell and (ii) exerts acytostatic or cytotoxic effect on a subpopulation of regulatory T cells;and (b) a pharmaceutically acceptable carrier.
 2. The method of claim 1,wherein the multivalent polypeptide comprises an antibody or fragmentsthereof.
 3. The method of claim 2, wherein the antibody is humanized orchimeric.
 4. The method of claim 2, wherein the antibody is a bispecificantibody.
 5. The method of claim 1, wherein the multivalent polypeptideis conjugated to a cytotoxic agent.
 6. The method of claim 5, whereinthe cytotoxic agent is a radionuclide.
 7. The method of claim 6, whereinthe radionuclide is selected from the group consisting of P³², P³³,Sc⁴⁷, Cu⁶⁴, Cu⁶⁷, As⁷⁷, Y⁹⁰, PH1⁰⁵, Pd¹⁰⁹, Ag¹¹¹, I¹²⁵, Pr¹⁴³, Sm¹⁵³,Tb¹⁶¹, Lu¹⁷⁷, Re¹⁸⁶, Re¹⁸⁸, Re¹⁸⁹, Ir¹⁹⁴, Au¹⁹⁹, Pb²¹², and Bi²¹³.